BACKGROUND: Wild-type RD114 virus is capable of generating syncytia during its replication, and it is believed that cell-free viruses direct the fusion of neighboring cells. The RD114 envelope (Env) that mediates this fusion event is now widely used to pseudotype retroviral and lentiviral vectors in gene therapy. Indeed, vectors pseudotyped with RD114 Env are very efficient to transfer genes into human hematopoietic cells, and they are resistant to human complement inactivation. In this study, we have tested the potential of RD114-pseudotyped vectors produced from the FLYRD18 packaging cell line to induce syncytia. METHODS: RD114-pseudotyped vectors produced from the FLYRD18 packaging cells were added on tumor cell lines, and the formation of syncytia was assessed by microscopy after cell fixation and methylene blue staining. The kinetics of syncytium formation was analyzed by time-lapse microscopy. Finally, the cytotoxic effect of RD114-pseudotyped vectors was measured by the MTT assay on tumor cells, and in combination with the TK/GCV strategy. RESULTS: We have found that these vectors were able to mediate cell-to-cell fusion of human tumor cell lines. A few hours after addition of the vector, cells started to aggregate to form syncytia that eventually evolved toward cell death 48 h postinfection. RD114-pseudotyped vectors were very efficient at killing human cancer cells, and they were also able to enhance dramatically the cytotoxic effect of the TK/GCV strategy. CONCLUSIONS: These findings indicate that RD114-pseudotyped vectors used alone, or in combination with a suicide gene therapy approach, have great potential for the treatment of cancer.